Primary open-angle glaucoma (POAG) is a common disease, particularly with advancing age, and requires chronic medication and/or surgical intervention to avert serious vision Loss. No cure or prevention is available and the molecular etiology of this blinding disease remains unclear. Obstruction of the aqueous outflow pathway at the level of the trabecular meshwork (TM) results in elevated intraocular pressure (IOP), frequently culminating in optic nerve damage. The normal trabecular resistance to aqueous humor outflow is thought to be provided by extracellular matrix macromolecules, particularly by the highly-charged glycosaminoglycan (GAG) side-chains of one or more trabecular proteoglycan. Disruption of the metabolism of these proteoglycans is a likely candidate in the development of this disease. No authenticated POAG culture or animal model systems has been found or developed. This proposal is designed to test the hypothesis that reducing the expression of trabecular stromelysin (proteoglycanase), a member of the matrix metalloproteinase family, or increasing the expression of the tissue inhibitor (TIMP) will diminish proteoglycan turnover and increase trabecular outflow resistance, i.e. cause glaucoma. Basic kinetic and binding studies will be conducted with recombinant stromelysin and TIMP produced in E. coli and in a mammalian retroviral protein, expression system to clarify details of their behavior and interactions. These proteins will then be overexpressed in trabecular cells by transfection with mammalian expression plasmids containing their cDNAs and by virion infection using engineered retrovirus containing their cDNAs. Underexpression in trabecular cells will be achieved by exposure to antisense oligonucleotides designed to block protein translation from their mRNAs. These expression and antisense studies will be conducted with human fibroblast and TM cells in culture to define their molecular behavior and then with TM cells in the perfused explant organ culture system. This organ culture system will be used to measure changes in proteoglycan turnover and then to evaluate the effects on aqueous outflow facility, thereby testing the hypothesis. A positive outcome to this test will produce the first real POAG model system for testing cures, preventions and treatments of this disease. In addition this will provide very strong evidence supporting a stromelysin disfunction in the molecular etiology of POAG.